Nucleotides encoding stop codons in multiple reading frames and methods of use

ABSTRACT

Compositions having polynucleotides encoding multiple translational stop signals in more than one reading frame are provided. The compositions include isolated polynucleotides, expression cassettes, and vectors, as well as host cells, prokaryotic organisms, and eukaryotic organisms comprising the polynucleotide(s). Methods include using the polynucleotides to stop translation of an mRNA into a protein, to produce a transformed cell and/or organism comprising the polynucleotide, and to identify transformed cells or organisms of a specific lineage.

CROSS-REFERENCE TO RELATED APPLICATIONS

This continuation application claims the benefit of U.S. Utilityapplication Ser. No. 12/183,135 filed Jul. 31, 2008 and U.S. ProvisionalApplication Ser. No. 60/953,698 filed Aug. 3, 2007, both of which areherein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to molecular biology and gene expression.

BACKGROUND

During transcription, transcript termination may not take place exactlyat the termination signal at a low, yet significant frequency.Incomplete and/or inaccurate transcription termination may producemessenger RNA (mRNA) transcripts with additional nucleotides containingopen reading frames (ORFS) that may be translated to produce unintendedproteins. During the production of transgenic cells and organisms,random insertion of the polynucleotide of interest may produce spuriousopen reading frames (ORFS) at the insertion locus, which may produce anunintended protein product. Various oversight agencies may haveregulatory concerns regarding any unintended protein products.Researchers and companies strive to develop and select transgenicorganisms products that do not produce or contain unintended products.Therefore, there is a need for compositions and methods to bettercontrol, eliminate, and/or minimize the production and/or accumulationof any unintended protein(s) in a transgenic organism.

SUMMARY

Compositions having polynucleotides encoding multiple translational stopsignals in more than one reading frame are provided. The compositionsinclude isolated polynucleotides, expression cassettes, and vectors, aswell as host cells, prokaryotic organisms, and eukaryotic organismscomprising the polynucleotide(s). Methods include using thepolynucleotides to stop translation of an mRNA into a protein, toproduce a transformed cell and/or organism comprising thepolynucleotide, and to identify transformed cells or organisms of aspecific lineage.

BRIEF DESCRIPTION OF FIGURES

FIG. 1. Several examples of ALLSTOPS polynucleotides are shown havingstop codons in all six reading frames. The stop codons are shown in boldtext.

DETAILED DESCRIPTION

The present invention now will be described more fully hereinafter withreference to the accompanying examples, in which some, but not all ofthe possible variations encompassed by the teachings are shown. Thisinvention may be embodied in different forms and should not be construedas limited solely to the variations set forth herein to satisfyapplicable legal requirements. Modifications and variations could beenvisioned by one of skill in art, and are therefore included within thescope of the appended claims. The disclosure of each reference set forthherein is incorporated by reference in its entirety. Although specificterms are employed within, they are used in a generic and descriptivesense only and not for purposes of limitation. The articles “a” and “an”as used herein to refer to at least one, for example “an element” meansat least one of the element.

Isolated polynucleotides encoding stop codons in multiple reading frames(ALLSTOPS) are provided. In some examples the isolated polynucleotidesencode stop codons in all reading frames in one orientation of atranscript. In some examples the isolated polynucleotides encode stopcodons in at least one reading frame in both possible transcriptorientations. In some examples the isolated polynucleotides encode stopcodons in all six possible reading frames of a transcript. In someexamples the ALLSTOPS polynucleotide is at least 12 nucleotides long. Insome examples the ALLSTOPS polynucleotide is at least 12 nucleotides toabout 450 nucleotides long. In some examples the ALLSTOPS polynucleotidedoes not encode a functional polypeptide. In some examples the ALLSTOPSpolynucleotide is a polynucleotide substantially similar to apolynucleotide of SEQ ID NOS: 1-7. In some examples the ALLSTOPSpolynucleotide comprises a functional fragment of a polynucleotide ofSEQ ID NOS: 1-7, which retains the encoded stop codons. In some examplesthe ALLSTOPS polynucleotide is a polynucleotide comprising SEQ ID NOS:1-7. In some examples the isolated polynucleotide comprises more thanone ALLSTOPS polynucleotide. In some examples the isolatedpolynucleotide comprises more than one ALLSTOPS polynucleotide, whereinat least one ALLSTOPS polynucleotide is substantially similar to any oneof SEQ ID NOS: 1-7. In some examples the isolated polynucleotidecomprises more than one ALLSTOPS polynucleotide, wherein at least oneALLSTOPS polynucleotide encoding is any one of SEQ ID NOS: 1-7.

Expression cassettes, DNA constructs, and vectors comprising at leastone ALLSTOPS polynucleotide are provided. In some examples, theexpression cassette, DNA construct, or vector is a T-DNA. In someexamples, the expression cassette, DNA construct, or vector is a viralDNA, including a viral replicon. In some examples, the expressioncassette, DNA construct or vector further comprises a polynucleotide ofinterest (POI). In some examples the polynucleotide of interest isoperably linked to at least one ALLSTOPS polynucleotide. In someexamples the polynucleotide of interest is operably linked to at leastone promoter (PRO) functional in a host cell. In some examples thepolynucleotide of interest is operably linked to at least one promoterand further operably linked to at least one ALLSTOPS polynucleotide. Insome examples a polynucleotide of interest is operably linked on bothsides to an ALLSTOPS polynucleotide. In some examples, the expressioncassette, DNA construct, or vector further comprises more than onepolynucleotide of interest, wherein each polynucleotide of interest isoptionally operably linked to a promoter. When the expression cassette,DNA construct, or vector comprises more than one polynucleotide ifinterest, an ALLSTOPS polynucleotide can be positioned outside of allpolynucleotides of interest, between polynucleotides of interest orbetween only select pairs or clusters of polynucleotides of interest orany combination thereof. Numerous configurations are possible, includingbut not limited to examples such as: ALLSTOPS::POI1-POI2;ALLSTOPS::POI1-POI2::ALLSTOPS; ALLSTOPS::PRO1::POI1-POI2;ALLSTOPS::PRO1::POI1-POI2::ALLSTOPS; ALLSTOPS::POI1-PRO2::POI2;ALLSTOPS::POI1-PRO2::POI2::ALLSTOPS; ALLSTOPS::PRO1::POI1-PRO2::POI2;ALLSTOPS::PRO1::POI1-PRO2::POI2::ALLSTOPS; ALLSTOPS::POI1-POI2::PRO2;ALLSTOPS::POI1-POI2::PRO2::ALLSTOPS; ALLSTOPS::POI1::ALLSTOPS-POI2;ALLSTOPS::POI1::ALLSTOPS::POI2::ALLSTOPS or combinations thereof.

Expression cassettes, DNA constructs, and vectors comprising at leastone ALLSTOPS polynucleotide may optionally comprise otherpolynucleotides including screenable markers, promoters, enhancers,terminators, untranslated regions, insulators, multiple cloning sites,restriction sites, homing endonuclease sites, recombination sites,transposition sequences, linkers, adapters, other sequences or anycombination thereof.

Host cells comprising at least one ALLSTOPS polynucleotide are provided.Host cells include prokaryotes, viruses, and eukaryotes. Prokaryotesinclude bacteria such as E. coli or Agrobacterium, including bacteriaused to propagate, amplify, express and/or transfer polynucleotides toanother host. Viruses include phage, plant viruses, avian viruses, andmammalian viruses, including viruses used to propagate, amplify, expressand/or transfer polynucleotides to another host. Eukaryotes includeyeast, fungi, plants, and animal cells. Host organisms comprising a hostcell having an ALLSTOPS polynucleotide are also provided. In someexamples the host cell is a plant cell. In some examples the plant cellis from a monocot, including but not limited to maize, barley, wheat,oat, rye, millet, sorghum, rice, switchgrass or turfgrass. In someexamples the plant cell is from a dicot, including but not limited tosoy, Brassica, alfalfa, Arabidopsis, tobacco, sunflower, or safflower.Seeds comprising an ALLSTOPS polynucleotide are also provided.

Methods using the isolated ALLSTOPS polynucleotide compositions areprovided. Methods include using the isolated polynucleotides to producea transformed host cell having the polynucleotide inserted into itsgenome. The ALLSTOPS polynucleotide may be inserted into a nuclear,organellar and/or plastidic genome. In some examples, insertion of anALLSTOPS polynucleotide into a host genome truncates translation of anyunintended mRNA transcript. In some examples, insertion of an ALLSTOPSpolynucleotide into a host genome stimulates degradation of anyunintended mRNA transcript. In some examples, insertion of an ALLSTOPSpolynucleotide into a host genome provides a means to identifytransformed cells, organisms and any progeny derived from thetransformed host cell. In some examples, the progeny comprise aproprietary germplasm or derivative thereof. Any host cell can be usedin the methods, including but not limited to bacterial cells, viruses,plant cells and mammalian cells. In some examples the host cell is abacterial cell, including but not limited to E. coli or Agrobacterium.In some examples the host cell is a plant cell. In some examples theplant cell is from a monocot, including but not limited to maize,barley, wheat, oat, rye, millet, sorghum, rice, switchgrass orturfgrass. In some examples the plant cell is from a dicot, includingbut not limited to soy, Brassica, alfalfa, Arabidopsis, tobacco,sunflower or safflower.

Transcriptional termination in eukaryotes is heterogeneous and thetermination sequences are not well-characterized. Additionally,unintended ORFs may occur due to the random integration of thetransgenic insertion into the genome of an organism. For example, ifinsertion occurs in the middle of an endogenous gene, the promoter fromthe endogenous gene could theoretically initiate transcription fromoutside the transgenic insert and transcribe into the transgenic insertgenerating an unintended transcript potentially containing an unintendedORF. In some instances, some rearrangement and or partial duplication ofthe transgene insert may occur, which may produce an ORF initiatingwithin the insert, which may extend into flanking genomic sequence.

Termination of protein biosynthesis occurs when the ribosome reaches astop codon, for which there is no tRNA. At this point, proteinbiosynthesis halts and a release factor binds to the stop codon. Bindingof the release factor induces a nucleophilic attack of the C-terminus ofthe nascent peptide by water and hydrolytic release of the peptide fromthe ribosome. The ribosome, release factor, and uncharged tRNAdissociate and translation is complete. All three stop codons, UAA, UAGand UGA, function in all living organisms. A single stop codon in theproper reading frame is sufficient to halt translation. Providing asingle stop codon in the correct frame and orientation would halttranslation of an unintended ORF. Therefore, even if an unintended mRNAtranscript is made, the production of an unintended protein is stoppedor minimized. Given the random nature of possible unintended ORFs, thecorrect frame and orientation of the ORF is not readily predictable,therefore providing stop codons in multiple reading frames andoptionally orientations assures terminating translation of spurioustranscripts. The combination of stop codons allows ALLSTOPS to be atranslational termination sequence capable of functioning in any readingframe. When ALLSTOPS is positioned downstream (3′) of any ORF,translation will be halted at the ALLSTOPS sequence.

In addition to stopping translation, ALLSTOPS can be used to identifytransformed cells or organisms. ALLSTOPS can be incorporated into everytransformed cell or organism generated and can be used to identifytransgenic events, proprietary germplasm, progeny and/or adventitiouspresence. Organisms can be analyzed to determine the presence or absenceof ALLSTOPS using standard methods including but not limited to Southernblots (Southern, (1975) Mol Biol 98:503-517), Northern blots, polymerasechain reaction (PCR and/or rtPCR) analyses, genomic sequencing, proteinprofile comparisons or any combination thereof.

A polynucleotide is any nucleic acid molecule comprising naturallyoccurring, synthetic, modified ribonucleotides, and/or modifieddeoxyribonucleotides and combinations thereof. Polynucleotides encompassall structural forms of sequences including, but not limited to,single-stranded, double-stranded, multi-stranded, linear, circular,branched, hairpins, stem-loop structures and the like.

ALLSTOPS polynucleotides include fragments and variants that retain thefunction of the ALLSTOPS polynucleotides disclosed herein. A fragment isa portion of the sequence which retains at least one function of theoriginal or reference sequence. Fragments of a polynucleotide sequenceincludes sequences which range from at least 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 100, 150, 200, 250, 300, 350, 400, 450nucleotides or longer. A variant sequence is a substantially similarsequence which retains at least one function of the original orreference sequence. For polynucleotides, a variant encompasses deletion,addition, and/or substitution of one or more nucleotides at one or moresites as compared to the original polynucleotide. For polypeptides, avariant encompasses deletion, addition, and/or substitution of one ormore amino acids at one or more sites as compared to the originalpolypeptide. Variants include sequences derived from an originalsequence. A substantially similar sequence, when aligned with anALLSTOPS sequence, has a significant number of nucleotides in common andretains the stop codons in multiple reading frames. Alternative stopcodons may be substituted, intervening nucleotides added, deleted orsubstituted or nucleotides deleted or added to either end of themolecule as long as the resulting polynucleotide retains stop codons inthe multiple reading frames. Any nucleotide can be substituted adjacentto or between stop codons, as long as the stop codons and reading framesare maintained. Due to the nature of ALLSTOPS polynucleotides,substantially similar sequences encompass sequences having a low percentsequence identity as compared to an ALLSTOPS sequence, but thesesubstantially similar sequences are readily identifiable by the numberof stop codons in multiple reading frames.

A DNA construct comprises an ALLSTOPS polynucleotide provided herein. Anexpression cassette, or recombinant expression cassette, comprises apolynucleotide which, when present in the genome of an organism, isheterologous or foreign to that chromosomal location in the host genome,wherein at least a portion of the expression cassette can provide orproduce at least one RNA. DNA constructs and expression cassette may beproduced using standard methods, see, for example, Sambrook, et al.,(1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.; and Glover (eds.)(1985) DNA Cloning: A Practical Approach, Volumes I and II. In preparingthe expression cassette or DNA construct, various fragments may bemanipulated to provide the sequences in a proper orientation and/or inthe proper reading frame. Adapters or linkers may be employed to jointhe fragments. Other manipulations may be used to provide convenientrestriction sites, remove of superfluous DNA, or remove of restrictionsites. For example, in vitro mutagenesis, primer repair, restriction,annealing, resubstitutions, transitions, transversions or recombinationsystems may be used.

Regulatory regions, including promoters, transcriptional regulatoryregions and/or translational termination regions, may be endogenous tothe host cell, genomic location, and/or to each other. Alternatively,the regulatory regions may be heterologous to the host cell, genomiclocation, and/or to each other. A heterologous sequence is a sequencethat originates from a foreign species, or, if from the same species, issubstantially modified from its native form in composition and/orgenomic locus by deliberate human intervention. An expression cassettemay include 5′ and 3′ regulatory sequences operably linked to apolynucleotide of interest, which is operably linked to an ALLSTOPSpolynucleotide. The ALLSTOPS polynucleotide may also be outside of theexpression cassette but still operably linked. Operably linked elementsmay be contiguous or non-contiguous. The expression cassette mayadditionally contain at least one additional polynucleotide of interestto be introduced into the organism. One or more expression cassettes maybe linked together as one transcriptional unit. In an Agrobacteriumvector for transformation of a plant cell, multiple expression cassettesmay be located in one transcriptional unit between the left and rightborders of the T-DNA vector. Alternatively, polynucleotide(s) ofinterest can be provided on separate DNA constructs or expressioncassettes. A DNA construct or expression cassette may optionally containa screenable marker.

The ALLSTOPS sequence may be positioned almost anywhere within a DNAconstruct. For example, the ALLSTOPS sequence could be positioned 3′ tothe transcriptional termination sequence, the translational terminationsequence, or to the polynucleotide of interest. The ALLSTOPS sequencemay also be located 5′ or 3′ to an expression cassette. Another optionis to place ALLSTOPS flanking both ends of the DNA construct. MultipleALLSTOPS sequences may be used within and/or outside of the expressioncassette. For example, the ALLSTOPS sequence could be placed betweeneach expression cassette within a transcriptional unit. Alternatively,ALLSTOPS could be places 5′ and 3′ to the expression cassette, in orderto prevent aberrant translation into the expression cassette or out ofthe expression cassette. An ALLSTOPS sequence could be placed either 3′or 5′ to the left border and an ALLSTOPS could be placed either 3′ or 5′to the right border in a T-DNA vector.

Any promoter, or combination of promoters, can be used. Promoters aretypically selected based on the desired expression profile. A promoteris a region of DNA involved in recognition and binding of RNA polymeraseand other proteins to initiate transcription. A plant promoter is apromoter capable of initiating transcription in a plant cell, for areview of plant promoters see, Potenza, et al., (2004) In Vitro Cell DevBiol 40:1-22.

Constitutive promoters include, for example, the core promoter of theRsyn7 promoter and other constitutive promoters disclosed in WO1999/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter(Odell, et al., (1985) Nature 313:810-812); rice actin (McElroy, et al.,(1990) Plant Cell 2:163-171); ubiquitin (Christensen, et al., (1989)Plant Mol Biol 12:619-632 and Christensen, et al., (1992) Plant Mol Biol18:675-689); pEMU (Last, et al., (1991) Theor Appl Genet. 81:581-588);MAS (Velten, et al., (1984) EMBO J. 3:2723-2730); ALS promoter (U.S.Pat. No. 5,659,026) and the like. Other constitutive promoters aredescribed, for example, in U.S. Pat. Nos. 5,608,149; 5,608,144;5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142 and6,177,611.

In some examples an inducible promoter may be used. Pathogen-induciblepromoters induced following infection by a pathogen include, but are notlimited to those regulating expression of PR proteins, SAR proteins,beta-1,3-glucanase, chitinase, etc. See, for example, Redolfi, et al.,(1983) Neth J Plant Pathol 89:245-254; Uknes, et al., (1992) Plant Cell4:645-656; Van Loon, (1985) Plant Mol Virol 4:111-116; WO 1999/43819;Marineau, et al., (1987) Plant Mol Biol 9:335-342; Matton, et al.,(1989) Mol Plant-Microbe Interact 2:325-331; Somsisch, et al., (1986)Proc Natl Acad Sci USA 83:2427-2430; Somsisch, et al., (1988) Mol GenGenet. 2:93-98; Yang (1996) Proc Natl Acad Sci USA 93:14972-14977; Chen,et al., (1996) Plant J 10:955-966; Zhang, et al., (1994) Proc Natl AcadSci USA 91:2507-2511; Warner, et al., (1993) Plant J 3:191-201;Siebertz, et al., (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386(nematode-inducible), and the references cited therein and Cordero, etal., (1992) Physiol Mol Plant Path 41:189-200 (Fusarium-inducible).Wound-inducible promoters include potato proteinase inhibitor (pin II)gene (Ryan (1990) Ann Rev Phytopath 28:425-449; Duan, et al., (1996) NatBiotechnol 14:494-498); wun1 and wun2 (U.S. Pat. No. 5,428,148); win1and win2 (Stanford, et al., (1989) Mol Gen Genet. 215:200-208); systemin(McGurl, et al., (1992) Science 225:1570-1573); WIP1 (Rohmeier, et al.,(1993) Plant Mol Biol 22:783-792; Eckelkamp, et al., (1993) FEBS Lett323:73-76); MPI gene (Corderok, et al., (1994) Plant J 6:141-150), andthe like.

Chemical-regulated promoters can be used to modulate the expression of agene through the application of an exogenous chemical regulator. Thepromoter may be a chemical-inducible promoter, where application of thechemical induces gene expression, or a chemical-repressible promoter,where application of the chemical represses gene expression.Chemical-inducible promoters include, but are not limited to, the maizeIn2-2 promoter, activated by benzenesulfonamide herbicide safeners (DeVeylder, et al., (1997) Plant Cell Physiol 38:568-77), the maize GSTpromoter (GST-II-27, WO 1993/01294), activated by hydrophobicelectrophilic compounds used as pre-emergent herbicides and the tobaccoPR-1a promoter (Ono, et al., (2004) Biosci Biotechnol Biochem 68:803-7)activated by salicylic acid. Other chemical-regulated promoters ofinterest include steroid-responsive promoters (see, for example, theglucocorticoid-inducible promoter in Schena, et al., (1991) Proc NatlAcad Sci USA 88:10421-10425 and McNellis, et al., (1998) Plant J14:247-257); tetracycline-inducible and tetracycline-repressiblepromoters (Gatz, et al., (1991) Mol Gen Genet. 227:229-237; U.S. Pat.Nos. 5,814,618 and 5,789,156).

Tissue-preferred promoters can be utilized to target enhanced expressionof a sequence of interest within a particular plant tissue.Tissue-preferred promoters include Kawamata, et al., (1997) Plant CellPhysiol 38:792-803; Hansen, et al., (1997) Mol Gen Genet. 254:337-343;Russell, et al., (1997) Transgenic Res 6:157-168; Rinehart, et al.,(1996) Plant Physiol 112:1331-1341; Van Camp, et al., (1996) PlantPhysiol 112:525-535; Canevascini, et al., (1996) Plant Physiol112:513-524; Lam (1994) Results Probl Cell Differ 20:181-196 andGuevara-Garcia, et al., (1993) Plant J 4:495-505. Leaf-preferredpromoters include, for example, Yamamoto, et al., (1997) Plant J12:255-265; Kwon, et al., (1994) Plant Physiol 105:357-67; Yamamoto, etal., (1994) Plant Cell Physiol 35:773-778; Gotor, et al., (1993) Plant J3:509-18; Orozco, et al., (1993) Plant Mol Biol 23:1129-1138; Matsuoka,et al., (1993) Proc Natl Acad Sci USA 90(20):9586-9590; cab and rubiscopromoters (Simpson, et al., (1958) EMBO J. 4:2723-2729; Timko, et al.,(1988) Nature 318:57-58). Root-preferred promoters are known andinclude, for example, Hire, et al., (1992) Plant Mol Biol 20:207-218(soybean root-specific glutamine synthase gene); Miao, et al., (1991)Plant Cell 3:11-22 (cytosolic glutamine synthase (GS) expressed in rootsand root nodules of soybean; Keller and Baumgartner, (1991) Plant Cell3:1051-1061 (root-specific control element in the GRP 1.8 gene of Frenchbean); Sanger, et al., (1990) Plant Mol Biol 14:433-443 (root-specificpromoter of A. tumefaciens mannopine synthase (MAS)); Bogusz, et al.,(1990) Plant Cell 2:633-641 (root-specific promoters isolated fromParasponia andersonii and Trema tomentosa); Leach and Aoyagi, (1991)Plant Sci 79:69-76 (A. rhizogenes roIC and roID root-inducing genes);Teeri, et al., (1989) EMBO J. 8:343-350 (Agrobacterium wound-inducedTR1′ and TR2′ genes); VfENOD-GRP3 gene promoter (Kuster, et al., (1995)Plant Mol Biol 29:759-772) and roIB promoter (Capana, et al., (1994)Plant Mol Biol 25(4):681-691; phaseolin gene (Murai, et al., (1983)Science 23:476-482; Sengopta-Gopalen, et al., (1988) Proc Natl Aced SciUSA 82:3320-3324). See also, U.S. Pat. Nos. 5,837,876; 5,750,386;5,633,363; 5,459,252; 5,401,836; 5,110,732 and 5,023,179. Seed-preferredpromoters include both seed-specific promoters active during seeddevelopment, as well as seed-germinating promoters active during seedgermination. See, Thompson, et al., (1989) BioEssays 10:108.Seed-preferred promoters include, but are not limited to, Cim1(cytokinin-induced message); cZ19B1 (maize 19 kDa zein) and milps(myo-inositol-1-phosphate synthase); (see, WO 2000/11177 and U.S. Pat.No. 6,225,529). For dicots, seed-preferred promoters include, but arenot limited to, bean β-phaseolin, napin, β-conglycinin, soybean lectin,cruciferin and the like. For monocots, seed-preferred promoters include,but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa gammazein, waxy, shrunken 1, shrunken 2, globulin 1, oleosin and nuc1. Seealso, WO 2000/12733, where seed-preferred promoters from end1 and end2genes are disclosed.

Commonly used prokaryotic control sequences include promoters fortranscription initiation, optionally with an operator, along withribosome binding sequences, include such commonly used promoters as thebeta lactamase (penicillinase) and lactose (lac) promoter systems(Chang, et al., (1977) Nature 198:1056), the tryptophan (trp) promotersystem (Goeddel, et al., (1980) Nucleic Acids Res 8:4057) and the lambdaderived P L promoter and N-gene ribosome binding site (Shimatake, etal., (1981) Nature 292:128).

The expression cassettes may additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders include picornavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein, et al., (1989)Proc Natl Acad Sci USA 86:6126-6130); polyvirus leaders, for example,TEV leader (Tobacco Etch Virus) (Gallie, et al., (1995) Gene165:233-238), MDMV leader (Maize Dwarf Mosaic Virus) (Allison, et al.,(1986) Virology 154:9-20; Kong, et al., (1988) Arch Virol143:1791-1799), and human immunoglobulin heavy-chain binding protein(BiP) (Macejak, et al., (1991) Nature 353:90-94); untranslated leaderfrom the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling,et al., (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV)(Gallie, et al., (1989) in Molecular Biology of RNA, ed. Cech (Liss, NewYork), pp. 237-256); and maize chlorotic mottle virus leader (MCMV)(Lommel, et al., (1991) Virology 81:382-385). See also, Della-Cioppa, etal., (1987) Plant Physiol 84:965-968. Other methods or sequences,including introns, known to enhance translation can also be utilizedalone or in combination with any of the 5′ leader sequences above.

Convenient transcriptional termination regions are available from theTi-plasmid of A. tumefaciens, such as the octopine synthase and nopalinesynthase (nos) termination regions. See also, Guerineau, et al., (1991)Mol Gen Genet. 262:141-144; Proudfoot, (1991) Cell 64:671-674; Sanfacon,et al., (1991) Genes Dev 5:141-149; Mogen, et al., (1990) Plant Cell2:1261-1272; Munroe, et al., (1990) Gene 91:151-158; Ballas, et al.,(1989) Nucleic Acids Res 17:7891-7903 and Joshi, et al., (1987) NucleicAcids Res 15:9627-9639.

A vector is selected to allow introduction into the appropriate hostcell. An expression cassette may be incorporated into a variety ofvectors. Vectors include circular or linear polynucleotides and can bederived from chromosomal, episomal and virus-derived vectors, including,mini-chromosomes, artificial chromosomes, satellite chromosomes and thelike; vectors derived from bacterial plasmids, from bacteriophage, fromyeast episomes, from yeast chromosomal elements, including yeastartificial chromosomes; from viruses, such as, baculoviruses,papovaviruses, such as, SV40, Vaccinia viruses, adenoviruses,poxviruses, pseudorabies viruses, retroviruses and plant viruses.Vectors may also be derived from combinations of these sources such asthose derived from plasmid and bacteriophage genetic elements, e.g.,cosmids and phagemids.

Bacterial vectors are typically of plasmid or phage origin. Appropriatebacterial cells are infected with phage vector particles or transfectedwith naked phage vector DNA. If a plasmid vector is used, the bacterialcells are transfected with the plasmid vector DNA. Prokaryotic/bacterialexpression systems for expressing a protein are available using Bacillussp. and Salmonella (Palva, et al., (1983) Gene 22:229-235; Mosbach, etal., (1983) Nature 302:543-545). The Tet operon and the Lac operon canbe used.

Two widely utilized yeasts for production of eukaryotic proteins areSaccharomyces cerevisiae and Pichia pastoris. Vectors, strains andprotocols for expression in Saccharomyces and Pichia are known andavailable from commercial suppliers (e.g., InVitrogen). Suitable vectorsusually have expression control sequences, such as promoters, including3-phosphoglycerate kinase or alcohol oxidase, and an origin ofreplication, termination sequences and the like as desired. Examples ofvectors for expression in yeast include pYepSec1 (Baldari, et al.,(1987) EMBO J. 6:229-234); pMFa (Kurjan, et al., (1982) Cell 30:933-943)and pJRY88 (Schultz, et al., (1987) Gene 54:113-123).

Polynucleotides can be expressed in insect cells using, for example,baculovirus expression vectors. Baculovirus vectors available forexpression of proteins in cultured insect cells, such as, Sf9 cells,include the pAc series (Smith, et al., (1983) Mol. Cell. Biol3:2156-2165) and the pVL series (Lucklow, et al., (1989) Virology170:31-39).

Established protocols with vectors and reagents are available fromcommercial suppliers (e.g., InVitrogen, Life Technologies Inc).Commercial vectors are available with various promoters, such aspolyhedrin and p10, optional signal sequences for protein secretion, oraffinity tags, such as 6× histidine. These recombinant viruses aregrown, maintained and propagated in commercially available cell linesderived from several insect species including Spodoptera frugiperda andTrichoplusia ni. The insect cells can be cultured using well-establishedprotocols in a variety of different media, for example, with and withoutbovine serum supplementation. The cultured cells are infected with therecombinant viruses and the sequence-of-interest is expressed. Proteinsexpressed with the baculovirus system have been extensivelycharacterized and, in many cases, their post-translational modificationssuch as phosphorylation, acylation, etc., are identical to the nativelyexpressed protein.

Polynucleotides may be expressed in mammalian cells using mammalianexpression vectors. Examples of mammalian expression vectors includepCDM8 (Seed, (1987) Nature 329:840) and pMT2PC (Kaufman, et. al., (1987)EMBO J 6:187-195).

DNA constructs can also comprise a screenable marker gene for theidentification and/or selection of transformed cells. Screenable markergenes can be used to identify and/or select transformed cells ortissues. Marker genes include genes encoding antibiotic resistance, suchas those encoding spectinomycin, ampicillin, kanamycin, tetracycline,Basta, neomycin phosphotransferase II (NEO) and hygromycinphosphotransferase (HPT), as well as genes conferring resistance toherbicidal compounds, such as glufosinate ammonium, bromoxynil,imidazolinones and 2,4-dichlorophenoxyacetate (2,4-D). Polynucleotideswhich encode products which are otherwise lacking in the cell can alsobe used as markers, including for example, tRNA genes, and auxotrophicmarkers. Additional markers include phenotypic markers such asβ-galactosidase, GUS and fluorescent proteins such as green fluorescentprotein (GFP) (Su, et al., (2004) Biotechnol Bioeng 85:610-9; Fetter, etal., (2004) Plant Cell 16:215-28), cyan fluorescent protein (CFP)(Bolte, et al., (2004) J Cell Science 117:943-54; Kato, et al., (2002)Plant Physiol 129:913-42), yellow fluorescent protein (YFP) (Bolte, etal., (2004) J Cell Science 117:943-54) and red fluorescent protein(RFP). Additional markers are available, see for example Yarranton,(1992) Curr Opin Biotech 3:506-511; Christopherson, et al., (1992) ProcNatl Acad Sci USA 89:6314-6318; Yao, et al., (1992) Cell 71:63-72;Reznikoff, (1992) Mol Microbiol 6:2419-2422; Barkley, et al., (1980) inThe Operon, pp. 177-220; Hu, et al., (1987) Cell 48:555-566; Brown, etal., (1987) Cell 49:603-612; Figge, et al., (1988) Cell 52:713-722;Deuschle, et al., (1989) Proc Natl Acad Sci USA 86:5400-5404; Fuerst, etal., (1989) Proc Natl Acad Sci USA 86:2549-2553; Deuschle, et al.,(1990) Science 248:480-483; Gossen, (1993) Ph.D. Thesis, University ofHeidelberg; Reines, et al., (1993) Proc Natl Acad Sci USA 90:1917-1921;Labow, et al., (1990) Mol Cell Biol 10:3343-3356; Zambretti, et al.,(1992) Proc Natl Acad Sci USA 89:3952-3956; Baim, et al., (1991) ProcNatl Acad Sci USA 88:5072-5076; Wyborski, et al., (1991) Nucleic AcidsRes 19:4647-4653; Hillenand-Wissman, (1989) Topics Mol Struc Biol10:143-162; Degenkolb, et al., (1991) Antimicrob Agents Chemother35:1591-1595; Kleinschnidt, et al., (1988) Biochemistry 27:1094-1104;Bonin, (1993) Ph.D. Thesis, University of Heidelberg; Gossen, et al.,(1992) Proc Natl Acad Sci USA 89:5547-5551; Oliva, et al., (1992)Antimicrob Agents Chemother 36:913-919; Hlavka, et al., (1985) Handbookof Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill,et al., (1988) Nature 334:721-724.

The methods herein involve introducing a polynucleotide encoding anALLSTOPS into a cell, tissue or organism. Introducing encompasses anymeans of presenting a composition to an organism in such a manner thatthe composition gains access to the interior of a cell of the organism.Compositions included polynucleotides, polypeptides, carriers, otherreagents, or combinations thereof. These methods do not depend on aparticular method for introducing a sequence into an organism, only thatthe polynucleotide or polypeptides gains access to the interior of atleast one cell of the organism.

Transformed host cells are prepared by introducing a DNA construct intothe cells using standard techniques. Methods for introducingpolynucleotide or polypeptides into organisms are known, including, butnot limited to, stable transformation methods, transient transformationmethods, virus-mediated methods and sexual crossing. Stabletransformation indicates that the introduced polynucleotide integratesinto the genome of the organism and is capable of being inherited byprogeny thereof. Transient transformation indicates that the introducedcomposition is only temporarily expressed or present in the organism.These methods include, but are not limited to, calcium phosphatetransfection, DEAE-dextran-mediated transfection, cationiclipid-mediated transfection, electroporation, transduction, infection,lipofection, Agrobacterium-mediated transformation, ballistic particleacceleration, and the like.

Protocols for introducing polynucleotides and polypeptides into plantsmay vary depending on the type of plant or plant cell targeted fortransformation, such as monocot or dicot. Suitable methods ofintroducing polynucleotides and polypeptides into plant cells andsubsequent insertion into the plant genome include microinjection(Crossway, et al., (1986) Biotechniques 4:320-334 and U.S. Pat. No.6,300,543), meristem transformation (U.S. Pat. No. 5,736,369),electroporation (Riggs, et al., (1986) Proc Natl Acad Sci USA83:5602-5606, Agrobacterium-mediated transformation (U.S. Pat. Nos.5,563,055 and 5,981,840), direct gene transfer (Paszkowski, et al.,(1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (U.S.Pat. Nos. 4,945,050; 5,879,918; 5,886,244; 5,932,782; Tomes, et al.,(1995) “Direct DNA Transfer into Intact Plant Cells via MicroprojectileBombardment,” in Plant Cell, Tissue, and Organ Culture: FundamentalMethods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe, etal., (1988) Biotechnology 6:923-926; Weissinger, et al., (1988) Ann RevGenet. 22:421-477; Sanford, et al., (1987) Particulate Science andTechnology 5:27-37 (onion); Christou, et al., (1988) Plant Physiol87:671-674 (soybean); Finer and McMullen, (1991) In Vitro Cell Dev Biol27P:175-182 (soybean); Singh, et al., (1998) Theor Appl Genet.96:319-324 (soybean); Datta, et al., (1990) Biotechnology 8:736-740(rice); Klein, et al., (1988) Proc Natl Acad Sci USA 85:4305-4309(maize); Klein, et al., (1988) Biotechnology 6:559-563 (maize); U.S.Pat. Nos. 5,240,855; 5,322,783 and 5,324,646; Klein, et al., (1988)Plant Physiol 91:440-444 (maize); Fromm, et al., (1990) Biotechnology8:833-839 (maize); Hooykaas-Van Slogteren, et al., (1984) Nature311:763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier, et al., (1987)Proc Natl Acad Sci USA 84:5345-5349 (Liliaceae); De Wet, et al., (1985)in The Experimental Manipulation of Ovule Tissues, ed. Chapman, et al.,(Longman, N.Y.), pp. 197-209 (pollen); Kaeppler, et al., (1990) PlantCell Rep 9:415-418; Kaeppler, et al., (1992) Theor Appl Genet.84:560-566 (whisker-mediated transformation); D'Halluin, et al., (1992)Plant Cell 4:1495-1505 (electroporation); Li, et al., (1993) Plant CellRep 12:250-255; Christou and Ford, (1995) Annals of Botany 75:407-413(rice) and, Osjoda, et al., (1996) Nat Biotechnol 14:745-750 (maize viaAgrobacterium tumefaciens).

Alternatively, the polynucleotides may be introduced into plants bycontacting plants with a virus or viral nucleic acids. Generally, suchmethods involve incorporating a polynucleotide within a viral DNA or RNAmolecule. It is recognized that a polypeptide of interest may beinitially synthesized as part of a viral polyprotein, which later may beprocessed by proteolysis in vivo or in vitro to produce the desiredrecombinant protein. Useful promoters also encompass promoters utilizedfor transcription by viral RNA polymerases. Methods for introducingpolynucleotides into plants and expressing a protein encoded therein,involving viral DNA or RNA molecules, are known, see, for example, U.S.Pat. Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367 and 5,316,931.

Transient transformation methods include, but are not limited to, theintroduction of polypeptides directly into the organism, theintroduction of polynucleotides such as DNA and/or RNA polynucleotidesand the introduction of the RNA transcript, such as an mRNA into theorganism. Such methods include, for example, microinjection or particlebombardment. See, for example, Crossway, et al., (1986) Mol Gen Genet.202:179-185; Nomura, et al., (1986) Plant Sci 44:53-58; Hepler, et al.,(1994) Proc Natl Acad Sci USA 91:2176-2180 and Hush, et al., (1994) JCell Sci 107:775-784.

The cells having the introduced sequence may be grown into plants inaccordance with conventional ways, see, for example, McCormick, et al.,(1986) Plant Cell Rep 5:81-84. These plants may then be grown, selfpollinated, outcrossed or backcrossed and the resulting progeny havingthe polynucleotide and/or trait. Sexual crossing techniques include, butare not limited to, recurrent selection, mass selection, bulk selection,mass selection, backcrossing, pedigree breeding, open pollinationbreeding, restriction fragment length polymorphism enhanced selection,genetic marker enhanced selection, making double haploids andtransformation. Often combinations of these techniques are used. Two ormore generations may be grown to ensure that the polynucleotide isstably maintained and inherited, and seeds harvested. In this manner,transformed seed, also referred to as transgenic seed, having apolynucleotide are provided.

ALLSTOPS may be introduced into any cell from any organism. Examples ofsuch target cells include cells derived from vertebrates includingmammals, such as, humans, bovine species, ovine species murine species,simian species; fungi; bacteria; insect; plants and the like. Organismsof interest include, but are not limited to both prokaryotic andeukaryotic organisms including, for example, bacteria, viruses, yeast,insects, fungi, mammals and plants. The term plant includes plant cells,plant protoplasts, plant cell tissue cultures from which plants can beregenerated, plant calli, plant clumps and plant cells that are intactin plants or parts of plants such as embryos, pollen, ovules, seeds,flowers, kernels, ears, cobs, husks, stalks, roots, root tips, anthersand the like.

ALLSTOPS may be introduced into any plant species, including, but notlimited to, monocots and dicots. Examples of plant species of interestinclude, but are not limited to, corn (Zea mays), Brassica sp. (e.g., B.napus, B. rapa, B. juncea), Brassica species useful as sources of seedoil, alfalfa (Medicago sativa), rice (Otyza sativa), rye (Secalecereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g.,pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum),foxtail millet (Setaria italica), finger millet (Eleusine coracana)),sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat(Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum),potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton(Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoeabatatus), cassaya (Manihot esculenta), coffee (Coffea spp.), coconut(Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrusspp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musaspp.), avocado (Persea americana), fig (Ficus casica), guava (Psidiumguajava), mango (Mangifera indica), olive (Olea europaea), papaya(Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamiaintegrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris),sugarcane (Saccharum spp.), turfgrass, switchgrass, oats, barley,vegetables, ornamentals, and conifers. Vegetables include tomatoes(Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans(Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrusspp.), and members of the genus Cucumis such as cucumber (C. sativus),cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamentalsinclude azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea),hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipaspp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation(Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), andchrysanthemum. Conifers include, for example, pines such as loblollypine (Pinus taeda), slash pine (Pinus effiotii), ponderosa pine (Pinusponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinusradiata); Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsugacanadensis); Sitka spruce (Picea glauca); redwood (Sequoiasempervirens); true firs such as silver fir (Abies amabilis) and balsamfir (Abies balsamea); and cedars such as Western red cedar (Thujaplicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).

Prokaryotic cells may also be used. Prokaryotes include various strainsof E. coli; however, other microbial strains may also be used,including, for example, Bacillus sp, Salmonella and Agrobacterium.Exemplary Agrobacterium strains include C58c1 (pGUSINT), Agt121(pBUSINT), EHA101 (pMTCA23GUSINT), EHA105 (pMT1), LBA4404 (pTOK233),GU2260, BU3600, AGL-1 and LBA4402. Such strains are described in detailin Chan, et al., (1992) Plant Cell Physiol 33:577; Smith, et al., (1995)Crop Sci 35:301 and Hiei, et al., (1994) Plant J 6:271-282. Exemplarybacterial strains include, but are not limited to, C600 (ATCC 23724),C600hfl, DH1 (ATCC 33849), DH5α, DH5αF′, ER1727, GM31, GM119 (ATCC53339), GM2163, HB101 (ATCC 33694), JM83 (ATCC 35607), JM101 (ATCC33876), JM103 (ATCC 39403), JM105 (ATCC 47016), JM107 (ATCC 47014),JM108, JM109 (ATCC53323), JM110 (ATCC 47013), LE392 (ATCC 33572), K802(ATCC 33526), NM522 (ATCC 47000), RR1 (ATCC31343), X¹⁹⁹⁷ (ATCC 31244)and Y1088 (ATCC 37195). See also, Jendrisak, et al., (1987) Guide toMolecular Cloning Techniques, Academic Press, 359-371, Hanahan, et al.,(1983) J Mol Biol 166:557-580, Schatz, et al., (1989) Cell 59:1035,Bullock, et al., (1987) BioTechniques 5:376-378, ATCC Bacteria andBacteriophages (1996) 9th Edition, and Palmer, et al., (1994) Gene143:7-8.

Viral strains include, but are not limited to, geminivirus, begomovirus,curtovirus, mastrevirus, (−) strand RNA viruses, (+) strand RNA viruses,potyvirus, potexvirus, tobamovirus or other DNA viruses, nanoviruses,viroids and the like, for example, African cassaya mosaic virus (ACMV)(Ward, et al., (1988) EMBO J. 7:899-904 and Hayes, et al., (1988) Nature334:179-182), barley stripe mosaic virus (BSM) (Joshi, et al., (1990)EMBO J. 9:2663-2669), cauliflower mosaic virus (CaMV) (Gronenborn, etal., (1981) Nature 294:773-776 and Brisson, et al., (1984) Nature310:511-514), maize streak virus (MSV) (Lazarowitz, et al., (1989) EMBOJ. 8:1023-1032 and Shen, et al., (1994) J Gen Virol 76:965-969), tobaccomosaic virus (TMV) (Takamatsu, et al., (1987) EMBO J. 6:307-311 andDawson, et al., (1989) Virology 172:285-292), tomato golden mosaic virus(TGMV) (Elmer, et al., (1990) Nucleic Acids Res 18:2001-2006) and wheatdwarf virus (WDV) (Woolston, et al., (1989) Nucleic Acids Res17:6029-6041) and derivatives thereof. See also, Porat, et al., (1996)Mol Biotechnol 5:209-221.

Example 1

Any method can be used to design a polynucleotide encoding stop codonsin multiple reading frames. In this example, the polynucleotidesencoding stop codons in multiple reading frames were designed manually.These polynucleotides can optionally be designed to include otherelements, such as sequences to facilitate isolation, identification,manipulation and/or cloning of the polynucleotide or a cell comprisingthe polynucleotide and include elements such as restriction enzymerecognition sites, homing endonuclease recognition sites, multiplecloning sites, transposition elements, recombination sites or anycombination thereof.

ALLSTOPS polynucleotides were designed by selecting a stop codon, addingone or more nucleotides to shift the reading frame, selecting anotherstop codon and repeating these steps until the designed polynucleotidehad stop codons in the desired reading frames. Depending on the stopcodons selected, double-stranded sequences as short as 12 bp can bedesigned having stop codons in all 6 possible reading frames.

To produce an ALLSTOPS polynucleotide comprising SEQ ID NO: 1 and havingto suitable restriction sites for cloning into a T-DNA target vector RBregion, four oligonucleotides for were designed and synthesized (SEQ IDNOS: 8-11). The oligonucleotides were annealed, ligated into linearvectors, and sequenced. A polynucleotide comprising ALLSTOPS sequence(SEQ ID NO: 1) was inserted into a T-DNA vector, approximately 250 bpdownstream (3′ to) the T-DNA right border (RB) sequence.

To produce an ALLSTOPS polynucleotide comprising SEQ ID NO: 1 and havingto suitable restriction sites for cloning into a T-DNA target vector LBregion, four oligonucleotides for were designed and synthesized (SEQ IDNOS: 12-15). The oligonucleotides were annealed, ligated into linearvectors, and sequenced. A polynucleotide comprising ALLSTOPS sequence(SEQ ID NO: 1) was inserted into the T-DNA vector, approximately 150 bpupstream (5′ to) the T-DNA left border (LB) sequence.

Synthetic oligonucleotides were designed and can be chemicallysynthesized to generate the double-stranded DNA sequence of SEQ ID NOS:2-7. The oligonucleotides are annealed, ligated into linear vectors andsequenced.

In addition, ALLSTOPS sequences can be positioned between geneexpression cassettes in stacked gene constructs to interrupt anyunintended ORFs arising in one cassette and extending into an adjacentcassette.

Besides including an ALLSTOPS polynucleotide adjacent to or flankingboth sides of an expression cassette in a DNA construct, an ALLSTOPSsequence can also be included in the middle of each arm of an invertedrepeat silencing cassette or be included in a DNA construct as aninverted repeat in its own right. Including ALLSTOPS in an invertedrepeat structure may serve two purposes. Translation of any unintendedORFs in a silencing construct will be halted as for any placement ofALLSTOPS. In addition, it may be expected that siRNAs to the ALLSTOPSsequences will be generated by DICER or DICER-LIKE. Any siRNAs producedmay act to trigger the RISC-mediated degradation of any transcript thatcontains the ALLSTOPS sequence. In this way, unintended transcripts willbe degraded even before any translation occurs.

Example 2

Any transformation method can be used to deliver an ALLSTOPS containingpolynucleotide to any appropriate cell, organism, or tissue target. DNAconstructs for transformation comprising ALLSTOPS are produced usingstandard molecular biological techniques.

A. Particle Bombardment of Maize

Immature maize embryos from greenhouse or field grown High type II(Hill) donor plants are bombarded with an isolated polynucleotidecomprising an ALLSTOPs polynucleotide. If the polynucleotide does notinclude a selectable marker, another polynucleotide containing aselectable marker gene can be co-precipitated on the particles used forbombardment. For example, a plasmid containing the PAT gene (Wohlleben,et al., (1988) Gene 70:25-37) which confers resistance to the herbicideBialaphos can be used. Transformation is performed as follows.

The ears are surface sterilized in 50% Clorox bleach plus 0.5% Microdetergent for 20 minutes, and rinsed two times with sterile water.Immature embryos are excised and placed embryo axis side down (scutellumside up), 25 embryos per plate. These are cultured in the dark on 560 Lagar medium 4 days prior to bombardment. Medium 560 L is an N6-basedmedium containing Eriksson's vitamins, thiamine, sucrose, 2,4-D, andsilver nitrate. The day of bombardment, the embryos are transferred to560Y medium for 4 hours and are arranged within a 2.5-cm target zone.Medium 560Y is a high osmoticum medium (560 L with high sucroseconcentration).

A circular or linear DNA construct comprising an ALLSTOPS sequence, andoptionally a polynucleotide of interest operably linked to a promoter,is constructed. This DNA construct, optionally plus a DNA constructcontaining a PAT selectable marker if needed, is precipitated onto 1.0μm (average diameter) gold pellets using a CaCl₂ precipitation procedureas follows: 100 μl prepared gold particles (0.6 mg) in water, 20 μl (2μg) DNA in TrisEDTA buffer (1 μg total), 100 μl 2.5 M CaCl₂, 40 μl 0.1 Mspermidine.

Each reagent is added sequentially to the gold particle suspension. Thefinal mixture is sonicated briefly. After the precipitation period, thetubes are centrifuged briefly, liquid removed, washed with 500 μl 100%ethanol, and centrifuged again for 30 seconds. Again the liquid isremoved, and 60 μl 100% ethanol is added to the final gold particlepellet. For particle gun bombardment, the gold/DNA particles are brieflysonicated and 5 μl spotted onto the center of each macrocarrier andallowed to dry about 2 minutes before bombardment.

The sample plates are bombarded at a distance of 8 cm from the stoppingscreen to the tissue, using a DuPont biolistics helium particle gun. Allsamples receive a single shot at 650 PSI, with a total of ten aliquotstaken from each tube of prepared particles/DNA.

Four to 12 hours post bombardment, the embryos are moved to a lowosmoticum callus initiation medium for 3-7 days, then transferred toselection medium 3 mg/liter Bialaphos, and subcultured every 2 weeks.After approximately 10 weeks of selection, callus clones are sampled forPCR and/or activity of the polynucleotide of interest. Positive linesare transferred to regeneration medium to initiate plant regeneration.Following somatic embryo maturation (2-4 weeks), well-developed somaticembryos are transferred to medium for germination and transferred to alighted culture room. Approximately 7-10 days later, developingplantlets are transferred to medium in tubes for 7-10 days untilplantlets are well established. Plants are then transferred to insertsin flats (equivalent to 2.5″ pot) containing potting soil and grown for1 week in a growth chamber, subsequently grown an additional 1-2 weeksin the greenhouse, then transferred to Classic™ 600 pots (1.6 gallon)and grown to maturity. Plants are monitored for expression of thepolynucleotide of interest.

B. Agrobacterium-Mediated Transformation of Maize

For Agrobacterium-mediated transformation of maize, a polynucleotidecomprising a recombination site, transfer cassette, target site, and/orrecombinase provided herein is used with the method of Zhao (U.S. Pat.No. 5,981,840).

Briefly, immature embryos are isolated from maize and the embryoscontacted with a suspension of Agrobacterium containing a polynucleotideof interest, where the bacteria are capable of transferring thenucleotide sequence of interest to at least one cell of at least one ofthe immature embryos (step 1: the infection step). In this step theimmature embryos are immersed in an Agrobacterium suspension for theinitiation of inoculation. The embryos are co-cultured for a time withthe Agrobacterium (step 2: the co-cultivation step). Following thisco-cultivation period an optional “resting” step may be performed (step3: resting step). The immature embryos are cultured on solid medium withantibiotic, but without a selecting agent, for elimination ofAgrobacterium and for a resting phase for the infected cells. Next,inoculated embryos are cultured on medium containing a selective agentand growing transformed callus is recovered (step 4: the selectionstep). The immature embryos are cultured on solid medium with aselective agent resulting in the selective growth of transformed cells.The callus is then regenerated into plants (step 5: the regenerationstep), and calli grown on selective medium are cultured on solid mediumto regenerate the plants.

C. Particle Bombardment Transformation of Soy

A polynucleotide comprising an ALLSTOPS polynucleotide, and optionally apolynucleotide of interest operably linked to a promoter, can beintroduced into embryogenic suspension cultures of soybean by particlebombardment using essentially the methods described in Parrott, et al.,(1989) Plant Cell Rep 7:615-617. This method, with modifications, isdescribed below.

Seed is removed from pods when the cotyledons are between 3 and 5 mm inlength. The seeds are sterilized in a bleach solution (0.5%) for 15minutes after which time the seeds are rinsed with sterile distilledwater. The immature cotyledons are excised by first cutting away theportion of the seed that contains the embryo axis. The cotyledons arethen removed from the seed coat by gently pushing the distal end of theseed with the blunt end of the scalpel blade. The cotyledons are thenplaced in Petri dishes (flat side up) with SB1 initiation medium (MSsalts, B5 vitamins, 20 mg/L 2,4-D, 31.5 g/L sucrose, 8 g/L TC Agar, pH5.8). The Petri plates are incubated in the light (16 hr day; 75-80 μE)at 26° C. After 4 weeks of incubation the cotyledons are transferred tofresh SB1 medium. After an additional two weeks, globular stage somaticembryos that exhibit proliferative areas are excised and transferred toFN Lite liquid medium (Samoylov, et al., (1998) In Vitro Cell Dev BiolPlant 34:8-13). About 10 to 12 small clusters of somatic embryos areplaced in 250 ml flasks containing 35 ml of SB172 medium. The soybeanembryogenic suspension cultures are maintained in 35 mL liquid media ona rotary shaker, 150 rpm, at 26° C. with florescent lights (20 μE) on a16:8 hour day/night schedule. Cultures are sub-cultured every two weeksby inoculating approximately 35 mg of tissue into 35 mL of liquidmedium.

Soybean embryogenic suspension cultures are then transformed usingparticle gun bombardment (e.g., Klein, et al., (1987) Nature 327:70;U.S. Pat. No. 4,945,050). A BioRad Biolisticä PDS1000/HE instrument canbe used for these transformations. A selectable marker gene, which isused to facilitate soybean transformation, is a chimeric gene composedof the 35S promoter from Cauliflower Mosaic Virus (Odell, et al., (1985)Nature 313:810-812), the hygromycin phosphotransferase gene from plasmidpJR225 (from E. coli; Gritz, et al., (1983) Gene 25:179-188) and the 3′region of the nopaline synthase gene from the T-DNA of the Ti plasmid ofAgrobacterium tumefaciens.

To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is agitated for three minutes, spun ina microfuge for 10 seconds and the supernatant removed. The DNA-coatedparticles are washed once in 400 μL 70% ethanol and resuspended in 40 μLof anhydrous ethanol. The DNA/particle suspension is sonicated threetimes for one second each. Five μL of the DNA-coated gold particles arethen loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. Membrane rupture pressure is set at 1100 psi andthe chamber is evacuated to a vacuum of 28 inches mercury. The tissue isplaced approximately 8 cm away from the retaining screen, and isbombarded three times. Following bombardment, the tissue is divided inhalf and placed back into 35 ml of FN Lite medium.

Five to seven days after bombardment, the liquid medium is exchangedwith fresh medium. Eleven days post bombardment the medium is exchangedwith fresh medium containing 50 mg/mL hygromycin. This selective mediumis refreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue will be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line is treated as anindependent transformation event. These suspensions are then subculturedand maintained as clusters of immature embryos, or tissue is regeneratedinto whole plants by maturation and germination of individual embryos.

D. DNA Isolation from Plant Callus or Leaf Tissues

Putative transformation events can be screened for the presence of atransgene. Genomic DNA is extracted from calli or leaves using amodification of the CTAB (cetyltriethylammonium bromide, Sigma H5882)method described by Stacey and Isaac, (1994) In Methods in MolecularBiology Vol. 28, pp. 9-15, Ed. P. G. Isaac, Humana Press, Totowa, N.J.Approximately 100-200 mg of frozen tissue is ground into powder inliquid nitrogen and homogenized in 1 ml of CTAB extraction buffer (2%CTAB, 0.02 M EDTA, 0.1 M Tris-Cl pH 8, 1.4 M NaCl, 25 mM DTT) for 30 minat 65° C. Homogenized samples are allowed to cool at room temperaturefor 15 min before a single protein extraction with approximately 1 ml24:1 v/v chloroform:octanol is done. Samples are centrifuged for 7 minat 13,000 rpm and the upper layer of supernatant collected usingwide-mouthed pipette tips. DNA is precipitated from the supernatant byincubation in 95% ethanol on ice for 1 h. DNA threads are spooled onto aglass hook, washed in 75% ethanol containing 0.2 M sodium acetate for 10min, air-dried for 5 min and resuspended in TE buffer. Five μl RNAse Ais added to the samples and incubated at 37° C. for 1 h. Forquantification of genomic DNA, gel electrophoresis is performed using a0.8% agarose gel in 1×TBE buffer. One microliter of each of the samplesis fractionated alongside 200, 400, 600 and 800 ng μl-1λ uncut DNAmarkers.

E. Transformation of Bacterial Cells

The ALLSTOPS sequences provided herein can also be evaluated and used inbacterial cells, such as Agrobacterium or E. coli. Many commerciallyavailable competent cell lines and bacterial plasmids are well known andreadily available. Isolated polynucleotides for transformation andtransformation of bacterial cells can be done by any method known in theart. For example, methods of E. coli and other bacterial celltransformation, plasmid preparation, and the use of phages are detailed,for example, in Current Protocols in Molecular Biology (Ausubel, et al.,(eds.) (1994) a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc.). For example, an efficient electroporationprotocol (Tung and Chow, Current Protocols in Molecular Biology,Supplement 32, Fall 1995) is summarized below.

Inoculate 100 ml LB medium with 1 ml E. coli overnight culture. Incubateat 37° C. with vigorous shaking until culture reached OD600=0.6. Chillculture on ice 30 min, then pellet cells by centrifuging 4,000×g for 15min at 4° C. Wash cell pellet twice with 50 ml ice-cold 10% glycerol.After final wash, resuspend cell pellet to a final volume of 0.2 ml inice-cold GYT medium (10% v/v glycerol; 0.125% w/v yeast extract; 0.25%w/v tryptone). Electroporate in prechilled cuvettes using manufacturer'sconditions, for example 0.5 ng plasmid DNA/transformation using GenePulser (BioRad) set to 25 μF, 200 ohms, 2.5 kV. Immediately afterelectroporation, add 1 ml SOC medium and transfer cells to a culturetube. Incubate at 37° C. for 1 hr. Plate aliquots of cells on selectiveagar plates and incubate overnight at 37° C. Pick resistant colonies andarchive or grow to confirm DNA delivery and characterization of events.DNA can be isolated and analyzed from putative transformed lines usingany standard procedures, including commercially available kits.

F. Transformation of Yeast

The ALLSTOPS sequences provided herein can also be evaluated and used inyeast cells. Many commercially and/or publicly available strains of S.cerevisiae are available, as are plasmids used to transform these cells.For example, strains are available from the American Type CultureCollection (ATCC, Manassas, Va.) and includes the Yeast Genetic StockCenter inventory, which moved to the ATCC in 1998. Other yeast lines,such as S. pombe and P. pastoris, and the like are also available.Methods of yeast transformation, plasmid preparation, and the like aredetailed, for example, in Current Protocols in Molecular Biology(Ausubel, et al., (eds.) (1994) a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., see Unit 13 inparticular). Transformation methods for yeast include spheroplasttransformation, electroporation, and lithium acetate methods. Aversatile, high efficiency transformation method for yeast is describedby Gietz and Woods ((2002) Methods Enzymol 350:87-96) using lithiumacetate, PEG 3500 and carrier DNA.

G. Transformation of Mammalian Cells

The ALLSTOPS sequences provided herein can also be evaluated and used inmammalian cells, such as CHO, HeLa, BALB/c, fibroblasts, mouse embryonicstem cells and the like. Many commercially available competent celllines and plasmids are well known and readily available, for examplefrom the ATCC (Manassas, Va.). Isolated polynucleotides fortransformation and transformation of mammalian cells can be done by anystandard method. For example, methods of mammalian and other eukaryoticcell transformation, plasmid preparation and the use of viruses aredetailed, for example, in Current Protocols in Molecular Biology(Ausubel, et al., (eds.) (1994) a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., see, Unit 9, inparticular). For example, many methods are available, such as calciumphosphate transfection, electoporation, DEAE-dextran transfection,liposome-mediated transfection, and microinjection, as well as viraltechniques.

Example 3

The functionality of an ALLSTOPS polynucleotide can be confirmed invariety of ways. In one example a single vector is constructed for planttransformation wherein the construct has two expression cassettes: 1)ubi pro driving an inverted repeat of phytoene desaturase (PDS) withALLSTOPS incorporated into each arm and 2) H2B pro driving DS-RED withALLSTOPS added just after or in place of the natural stop codon as a 3′tag.

Control vectors for plant transformation are 1) ALLSTOPS-tagged DS-REDcassette paired with untagged PDS and 2) ALLSTOPS-tagged DS-RED pairedwith PDS tagged with something other than ALLSTOPS.

Plantlets transformed with the ALLSTOPS test construct should have thefollowing phenotypes:

green plantlets should be RFP+; and,

white/bleached plantlets should be RFP−.

Plantlets transformed with the control vectors should all be RFP+regardless of green/white phenotype.

Transient transformation can also be used to assay for ALLSTOPSfunction. In one example, constructs are designed which encode a fusionprotein with a GFP reporter with and without an intervening ALLSTOPS inthe linker region:

PRO::GUS-linker-GFP::term; and,

PRO::GUS-ALLSTOPS-GFP::term.

Transient transformation of E. coli, or other host cells, can be used tomeasure the relative expression of GUS and GFP from the construct withan intervening linker region as compared to the construct having theALLSTOPS linker.

1. An isolated polynucleotide encoding stop codons in all six readingframes, wherein the polynucleotide is from 12 to 450 nucleotides.
 2. Theisolated polynucleotide of claim 1, wherein the polynucleotide issubstantially identical to a polynucleotide of SEQ ID NO: 1-7.
 3. Theisolated polynucleotide of claim 1, wherein the polynucleotide isselected from the group consisting of SEQ ID NO: 1-7.
 4. A recombinantT-DNA expression cassette comprising a polynucleotide encoding stopcodons in all six reading frames, wherein the polynucleotide is from 12to 450 nucleotides.
 5. (canceled)
 6. A non-human host cell comprisingthe polynucleotide of any one of claims 1-3.
 7. The host cell of claim 6wherein the host cell is a plant cell.
 8. The host cell of claim 6wherein the host cell is an Agrobacterium
 9. A transformed plantcomprising the polynucleotide of any one of claims 1-3.
 10. Atransformed seed comprising the polynucleotide of any one of claims 1-3.11. The plant of claim 9 wherein the plant is a monocot or a dicot. 12.The plant of claim 11 wherein the plant is maize.
 13. A method toproduce a transformed host cell comprising: a) introducing a recombinantT-DNA expression cassette a polynucleotide encoding stop codons in allsix reading frames, wherein the polynucleotide is from 12 to 450nucleotides into the host cell; and, b) selecting a transformed hostcell having the polynucleotide inserted into its genome.
 14. The methodof claim 13, wherein insertion of the polynucleotide into the hostgenome truncates translation of any unintended messenger RNA transcript.15. The method of claim, wherein insertion of the polynucleotide intothe host genome stimulates degradation of any unintended messenger RNAtranscript.
 16. The method of claim 13, wherein insertion of thepolynucleotide into the host genome provides a means to identify anyprogeny derived from the transformed host cell.
 17. The method of claim16, wherein the progeny comprise proprietary germplasm.
 18. The methodof any one of claims 13-17 wherein the host cell is selected from thegroup consisting of a plant cell, a bacterial cell, or a mammalian cell.19. The method of any one of claims 13-18, wherein the host cell is aplant cell.
 20. The method of any one of claims 13-19, wherein the hostcell is a maize cell.
 21. The recombinant T-DNA expression cassette ofclaim 4, wherein the polynucleotide is substantially identical to apolynucleotide selected from the group consisting of: SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, andSEQ ID NO:
 7. 22. The recombinant T-DNA expression cassette of claim 4,wherein the polynucleotide is a polynucleotide of selected from thegroup consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO:
 7. 23. A method toproduce a transformed host cell of claim 13, wherein the polynucleotideis substantially identical to a polynucleotide selected from the groupconsisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO:
 7. 24. A method to produce atransformed host cell of claim 13, wherein the polynucleotide is apolynucleotide of selected from the group consisting of: SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,and SEQ ID NO: 7.